Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.
A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.
Henrick
K
K
National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom.
Bawumia
S
S
Barboni
E A
EA
Mehul
B
B
Hughes
R C
RC
D000595:Amino Acid Sequence; D000818:Animals; D000943:Antigens, Differentiation / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D005690:Galactose / Q000378:metabolism*; D037502:Galectin 3; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012794:Sialic Acids / Q000737:chemistry
D000943:Antigens, Differentiation; D017931:DNA Primers; D037502:Galectin 3; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D012794:Sialic Acids; D005690:Galactose